Mutations to transcription factor MAX allosterically increase DNA selectivity by altering folding and binding pathways

• Published in: Nature communications 2025-01, Vol.16 (1), p.636-17, Article 636
• Main Authors: Hastings, Renee, Aditham, Arjun K., DelRosso, Nicole, Suzuki, Peter H., Fordyce, Polly M.
• Format: Article
• Language: English
• Subjects: 49/47; 49/62; 49/79; 631/154/1435/2163; 631/45/612/1229; 631/57/2272/951; 82/80; Allosteric properties; Allosteric Regulation; Basic-Leucine Zipper Transcription Factors - chemistry; Basic-Leucine Zipper Transcription Factors - genetics; Basic-Leucine Zipper Transcription Factors - metabolism; Binding; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA - genetics; DNA - metabolism; Folding; Gene sequencing; Heterogeneity; Humanities and Social Sciences; Humans; Kinetics; Ligands; Microfluidics; Models, Molecular; multidisciplinary; Mutation; Nucleotide sequence; Nucleotides; Point mutation; Protein Binding; Protein engineering; Protein Folding; Proteins; Rate constants; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Science; Science (multidisciplinary); Selectivity; Thermodynamics; Transcription factors
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-55672-2

Understanding how proteins discriminate between preferred and non-preferred ligands (‘selectivity’) is essential for predicting biological function and a central goal of protein engineering efforts, yet the biophysical mechanisms underpinning selectivity remain poorly understood. Towards this end, we study how variants of the promiscuous transcription factor (TF) MAX ( H. sapiens ) alter DNA specificity and selectivity, yielding >1700  K d s and >500 rate constants in complex with multiple DNA sequences. Twenty-two of the 240 assayed MAX point mutations enhance selectivity, yet none of these mutations occur at residues that contact nucleotides in published structures. By applying thermodynamic and kinetic models to these results and previous observations for the highly similar yet far more selective TF Pho4 ( S. cerevisiae ), we find that these mutations enhance selectivity by altering partitioning between or affinity within conformations with different intrinsic selectivity, providing a mechanistic basis for allosteric modulation of ligand selectivity. These results highlight the importance of conformational heterogeneity in determining sequence selectivity and can guide future efforts to engineer selective proteins. In this work, high-throughput microfluidic assays reveal how allosteric substitutions that alter folding-and-binding pathways shape DNA specificity and selectivity landscapes in the human transcription factor MAX.